New resistance sources to root-knot nematode in Capsicum pepper

Thirty seven pepper genotypes, Capsicum chinense, C. annuum and C. frutescens, were characterized for resistance to three rootknot nematode species (Meloidogyne javanica, M. incognita race 1 and M. enterolobii) aiming to find potential resistance sources to be used in breeding programs. Three experiments were carried out, in 2013, 2014 and 2016, in a greenhouse, in randomized block design and six replicates, one plant/pot. Data were submitted to analysis of variance and average clustering. All evaluated pepper genotypes were resistant or immune to M. javanica. For M. incognita race 1, all accessions of C. frutescens, evaluated in the first experiment, were resistant, whereas the six accessions of C. chinense were susceptible. For M. incognita, all genotypes of C. chinense and C. annuum, evaluated in the second experiment, were resistant. In the third experiment, evaluating C. annuum genotypes, we verified that most were susceptible to M. incognita race 1; genotypes CNPH 30118 and CNPH 6144 were resistant, though. M. enterolobii, which is the most aggressive species, with few resistance sources described, showed a tendency of greater degree of resistance in C. chinense and C. frutescens. Although most of the evaluated genotypes were susceptible, significant differences were observed regarding the degree of susceptibility. The main contribution of this study was the identification of a genotype resistant to M. enterolobii, cultivar BRS Nandaia, which can be used in breeding programs.

problems for pepper cultivation is the root-knot nematode. These pathogens damage the plant root system and transport of water and nutrients, limiting its productivity (Kiewnick et al., 2009;Chaudhary & Kaul, 2012). P epper is grown in all Brazilian regions; however, no statistical analysis accurately quantifies the economic importance of this crop, since much of the production is sold in local and outdoor markets. One of the biggest Pofu, 2012; Hussain et al., 2013).
Another nematode species which has impaired vegetable production is M. enterolobii. This species was originally found in guava plants in 2001 in Pernambuco and Bahia States, and since then it is one of Brazilian producers' concern. In vegetables, this species was reported, for the first time, in São Paulo State, parasitizing bell pepper cv. 'Silver' rootstocks and 'Andréa' and 'Débora' tomato plants, resistant to M. incognita and M. javanica (Carneiro et al., 2006). Preliminary studies show that peppers of the genus Capsicum are more susceptible to M. enterolobii comparing to other species of root-knot nematodes (Kiewnick et al., 2009;Pinheiro et al., 2015). On the other hand, some reports on different levels of susceptibility or even resistance in pepper can be found in literature (Oliveira, 2007;Melo et al., 2011;Pinheiro et al., 2013;Gonçalves et al., 2014).
Due to high relevance of nematodes for pepper crop, the characterization of potential genotypes to become new cultivars or to be used for crossings to add resistance factor to other characters of agronomic importance is crucial. Therefore, this study aimed to characterize pepper genotypes in relation to resistance against three root-knot nematode species, allowing to select resistant inbred lines to be released as cultivars and/or as potential resistance sources for breeding programs.

MATERIAL AND METHODS
The experiments were carried out from April to July, 2013 -2016, and from October to January, 2014, in Distrito Federal, Brazil, in greenhouses.
The experiments were conducted in greenhouse in randomized block design in factorial scheme for each year, with six replicates, considering that the experimental unit consisted of one plant per pot with 1.5 liters sterilized substrate autoclaved at 121°C for 60 min. Sowings were done on April 24, 2013, October 23, 2014and April 18, 2016. About 20 days after sowing, plants were inoculated with suspension of 5,000 eggs and eventual second stage juveniles (J2) of each isolated species, in 5 mL water distributed around the stem base.
At 75 days after inoculation, eggs and J2 were extracted from the root system of the plants according to methodology of Boneti & Ferraz (1981). The following variables were evaluated: egg mass index (IMO): the root system was washed in running water, stained with Phloxin B solution 0.5 g/L water for 15 minutes. Then, the authors counted the number of egg masses of nematodes using a stereoscope microscope. IMO in roots was obtained according to Taylor & Sasser (1978), using a note scale from 0 to 5, in which 0 is related to roots without egg masses; 1 is related to 1 to 2 egg masses; 2 corresponds to a range from 3 to 10 egg masses; 3, from 11 to 30 egg masses; 4, from 31-100 egg masses and 5 corresponds to more than 100 egg masses/root system. The authors also evaluated gall index (IG), which is the number of galls in each root system in each plant/replicate. IG in roots was represented by the scale from 0 to 5, according to Taylor & Sasser (1978), replacing the quantification of egg masses by galls and then assigning the grades. To evaluate the number of eggs per gram of roots (eggs/g), roots were washed, dried at room temperature for five hours and weighed before being processed according to Bonetti & Ferraz (1981), counting the number of eggs of each plant divided by the weight, considering that in the experiment of 2013 this character was not evaluated. Reproduction factor (FR) was obtained dividing the final and initial population densities (FR=Pf/Pi), considering zero the immune value (I), lower than 1 resistant (R) and higher than 1 susceptible (S) (Oostenbrink, 1966).
Data eggs/g roots and FR were transformed in to meet the normal distribution. All data were submitted to analysis of individual and joint variance for three species of nematodes in each year, and average clustering by Scott-Knott, using Genes software (Cruz, 2013).  Taylor & Sasser (1978) where 0) roots without egg mass and/or galls; 1) roots with 1 to 2 egg masses and/or galls; 2) roots with 3 to 10 egg masses and/or galls; 3) roots with 11 to 30 egg masses and/or galls; 4) roots with 31 to 100 egg masses and/or galls; 5) roots with over 100 egg masses and/or galls. FR/reaction (reproduction factor, final population/initial population) according to Oostenbrink (1966) where I= immune (FR= 0); R= resistant (FR<1) and S= susceptible (FR>1). Averages followed by same letters do not differ from each other, Scott-Knott test (p<0.05). CV= coefficients of environmental variation. CVg/CV= genotypic and phenotypic coefficients of variation ratio. coefficients of variation showed lower value, and the coefficients of genotypic and environmental variation ratio (CVg/CV) were higher than the unit for most evaluated characters in the three experiments (Tables 1, 2 and 3). This result shows predominance of genetic variation, surpassing the environmental variation and, as a consequence, the Coefficients of environmental variation were higher for number of eggs per gram of roots (eggs/g), showing that this character is strongly influenced by environment, and that observations which were carried out using this variable would be unreliable (in the first experiment this character was not evaluated). For other characters, genotypes and nematode species was verified in the three experiments (2013, 2014 and 2016), showing that genotype response to resistance level was different depending on the evaluated nematode species. Significant differences were noticed in all analyses of individual variance (P<0.05) for all characters in both experiments (data not shown).

Significant interaction between
reliability of the estimates.
Oliveira (2007), evaluating different Capsicum species, observed that all nine C. chinense genotypes were susceptible to M. enterolobii, with FR lower than the ones obtained in C. annuum, though. He adds that the only C. frutescens genotype which was evaluated was  Taylor & Sasser (1978) where 0) roots without egg mass and/or galls; 1) roots with 1 to 2 egg masses and/or galls; 2) roots with 3 to 10 egg masses and/or galls; 3) roots with 11 to 30 egg masses and/or galls; 4) roots with 31 to 100 egg masses and/or galls; 5) roots with over 100 egg masses and/or galls. Eggs/g= number of eggs per gram of roots. FR/reaction (reproduction factor, final population/initial population) according to Oostenbrink (1966) where I= immune (FR= 0); R= resistant (FR<1) and S= susceptible (FR>1). Averages followed by same letters do not differ from each other, Scott-Knott test (p<0.05). CV= coefficients of environmental variation. CVg/CV= genotypic and phenotypic coefficients of variation ratio. All pepper genotypes evaluated in this study were resistant or immune to M. javanica. Four C. frutescens genotypes evaluated in the first experiment were resistant to M. incognita, whereas six C. chinense genotypes, in the same experiment, were susceptible. For M. incognita race 1, all C. chinense and C. annuum genotypes evaluated in the second experiment were resistant. In the third experiment, in which only C. annuum genotypes were evaluated, most were susceptible to M. incognita, but genotypes CNPH 30118 and CNPH 6144 were resistant. In relation to M. enterolobii, which is the most aggressive species, and few sources of resistance described in literature, a tendency of higher degree of resistance in C. chinense and C. frutescens was observed and, although most of the genotypes evaluated in the present study were susceptible, significant differences were observed regarding the degree of susceptibility. This information on different levels of resistance to root-knot nematodes among and in Capsicum species is of extreme importance for breeding programs, yet the main contribution of this study was the identification of resistance to M. enterolobii in 'BRS Nandaia', a C. chinense genotype. Nevertheless, further studies are necessary to investigate molecular bases of this resistance. IG (gall index) and IMO (egg mass) according to Taylor & Sasser (1978) where 0) roots without egg mass and/or galls; 1) roots with 1 to 2 egg masses and/or galls; 2) roots with 3 to 10 egg masses and/or galls; 3) roots with 11 to 30 egg masses and/or galls; 4) roots with 31 to 100 egg masses and/or galls; 5) roots with over 100 egg masses and/or galls. Eggs/g= number of eggs per gram of roots. FR/reaction (reproduction factor, final population/initial population) according to Oostenbrink (1966) where I= immune (FR= 0); R= resistant (FR<1) and S= susceptible (FR>1). Averages followed by same letters do not differ from each other, Scott-Knott test (p<0.05). CV= coefficients of environmental variation. CVg/CV= genotypic and phenotypic coefficients of variation ratio.